Method of isolating blue anthocyanin fractions

ABSTRACT

The present invention is directed to a method of isolating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts, or combinations thereof, at a select pH based on differences in polarity of the anthocyanin molecules in the anthocyanin-containing vegetable and fruit juices and extracts.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.14/773,103, filed Sep. 4, 2015, which is a 371 National Stage filing ofInternational Application Serial No. PCT/US2014/027319, filed Mar. 14,2014, which claims benefit of U.S. Provisional Patent Application Ser.No. 61/790,842, filed Mar. 15, 2013, all of which are herebyincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a method of obtaining natural blueanthocyanin-containing colorant compositions by selectively isolatingfractions of anthocyanin molecules from anthocyanin-containing vegetableand fruit juices and extracts.

Description of the Related Art

There is increasing interest in the food industry to replace syntheticmaterials for coloring foods with natural colorants.

One challenge in replacing synthetic colorants with natural colorantshas been identifying natural colorants that provide colorcharacteristics similar to those provided by synthetic colorants.

Natural colorants that provide the same color characteristics as thesynthetic blue colorant, FD&C Blue No. 1, have not been found, to thistime. The lack of appropriate natural cyan blue hue colorants has alsomade it challenging to obtain desired natural green hue colorants fromthe blending of natural blue and yellow colorants. Spirulina Blue, ablue-green algae-derived material, is used as a natural blue colorant,but does not provide the same color characteristics as FD&C Blue No. 1.

Anthocyanins are water-soluble compounds found in the cell vacuoles offruits, vegetables, and flower petals, and sometimes, roots, leaves,stems, and bracts of plants. At least in part due to their wideavailability, anthocyanin-containing vegetable and fruit juices andextracts have been used as natural, edible colorants and to producecolorant compositions, in particular, natural red, purple, and blue huecolorant compositions.

An anthocyanin comprises an anthocyanidin (the aglycone) esterified toone or more sugar molecules (the glycone(s)) to form a glycoside. Sugarmolecules may be attached at the C-3, C-5, C-7, C-3′, C-4′, and/or C-5′positions. Examples of sugar molecules found in anthocyanin structuresare arabinose, galactose, glucose, rhamnose, rutinose, sambubiose,sophorose, and xylose.

Anthocyanins may also be acylated, i.e., they may have one or moremolecules esterified to the sugar molecules, typically at the 6-positionof a monosaccharide, but also potentially at the 2-, 3-, or 4-positions.The most common acyl units include those derived from coumaric, ferulic,caffeic, sinapic, gallic, malonic, acetic, malic, succinic, vanillic,and oxalic acids.

The structure of an anthocyanidin is shown below in the flavylium cationform, which is the primary form under acidic conditions. Theanthocyanidin may be substituted with hydrogen, hydroxyl, and/ormethoxyl groups at various positions:

wherein R³ is H or OH,

R⁵ is H, OH, or OCH₃,

R⁶ is H or OH,

R⁷ is OH or OCH₃,

R^(3′) is H, OH, or OCH₃,

R^(4′) is OH or OCH₃, and

R^(5′) is H, OH, or OCH₃.

The most common anthocyanidins in nature are shown by the followingstructures:

Therefore, the class of compounds known as anthocyanins encompasses anenormous number of structurally diverse compounds based on differencesin primary structure, glycosylation and acylation patterns.

Known plant sources of anthocyanins include: (1) vegetables such as redcabbage, purple sweet potato, blue potato, red potato, red radish, blackcarrot, purple carrot, purple corn, red corn, red onion, purplebroccoli, red broccoli, purple cauliflower, rhubarb, black bean, redleaf lettuce, black rice and eggplant; and (2) fruits such asstrawberry, raspberry, cranberry, lingonberry, red grape, apple, blackcurrant, red currant, cherry, blueberry, elderberry, bilberry,crowberry, blackberry, chokeberry, gooseberry, açai, nectarine, peach,plum, blood orange and blue tomato. Each anthocyanin source containsdifferent amounts of multiple, distinct anthocyanin species, with 15 to30 structurally distinct anthocyanin molecules being common for a givenplant source.

The color characteristics of anthocyanin-containing vegetable and fruitjuices and extracts change as a result of changing pH.Anthocyanin-containing juices and extracts generally exhibit red hues atlow pH, and the hue shifts to purple as the pH is increased. Only a fewjuices and extracts exhibit a blue hue as pH is increased further.

The change in color of anthocyanin-containing juices and extractsresulting from changes in pH is related to the numerous secondarystructures of anthocyanins that may exist in equilibrium with theprimary flavylium cation structure in aqueous solution. When pH ischanged, the relative quantities of the different equilibrium structureswill change. At a given pH, one or more structural forms maypredominate, while others are present in low quantities or not present.For example, at very low pH, the flavylium cation form predominates. AspH is increased, molecules in the flavylium cation form may bedeprotonated and converted to the carbinol pseudobase form, which may befurther converted through loss of a water molecule and a proton to theneutral and ionized quinonoidal base forms, respectively, and further,to the chalcone form. These transformations reduce the quantity ofmolecules in the flavylium cation form and increase the quantities inthe other equilibrium forms to different extents. Therefore, thedifferent equilibrium structures exist in different relative quantitiesat higher pH compared to low pH. Each structural form of anthocyanin mayabsorb light differently, resulting in a different perceived color,including no color. Therefore, as the pH of the solution is changed,changes in the relative quantities of the different structural forms mayresult in changes in the color of the solution.

Each distinct anthocyanin molecule is characterized by its own set ofequilibrium molecular structures and equilibrium constants for thereactions that transform one structure into another. For example, thereaction transforming one anthocyanin equilibrium structure into anothermay have a particular acid dissociation constant, K_(a), associated withit. The reaction may also be discussed in terms of the logarithmicconstant, pK_(a), which is defined as −log₁₀ K_(a).

The flavylium cation and quinonoidal base structures have conjugatedbonds connecting all three rings of the anthocyanin molecules. Theextensive delocalized pi bonds allow the flavylium cation andquinonoidal base to absorb visible light, resulting in the perceived redhue of the flavylium cation at low pH and the purple or blue hue of theionized quinonoidal base at a higher pH. In contrast, the carbinolpseudobase and chalcone structures do not have delocalized pi bondsconnecting all three rings and are colorless or slightly yellow.

The substitution pattern of anthocyanins also affects color. Forexample, it is generally observed that the hue shifts from pink topurple when hydrogen atoms are replaced with hydroxyl groups. Similarly,the number of glycosyl (sugar) units and the number and type of acylunits are observed to affect color. However, these phenomena are notwell understood or predictable.

Additionally, intermolecular and intramolecular interactions also affectanthocyanin color. The same anthocyanin may produce different huesdepending on the other molecules present. For example, it is believedthat acyl groups on the anthocyanin sugars can fold in and protect theflavylium cation C-2 position from nucleophilic attack. Therefore, thisintramolecular interaction prevents formation of the colorless carbinolpseudobase structure. Similarly, it is believed that anthocyaninmolecules self-associate, which is evidenced by the fact that a two-foldincrease in anthocyanin concentration can cause a 300-fold increase inchroma, and can change the hue and value as well. It is hypothesizedthat this self-association is similar to intramolecular stacking, andprevents nucleophilic attack and formation of the carbinol pseudobasestructure.

Although it is known that factors such as pH, anthocyanin chemicalstructure, substituent patterns, inter- and intra-molecular interactionsall impact the color observed in anthocyanin-containing vegetable andfruit juices and extracts, it is not well understood how these factorsinteract to alter color; i.e., the specific cause and effect are notpredictable.

For example, individual anthocyanin molecules have been separated byHPLC, but the separation has always occurred at low pH, and the colorcharacteristics of individual anthocyanins were analyzed at low pH.Similarly, the effect of pH on the color characteristics ofanthocyanin-containing vegetable and fruit juices and extracts has beenstudied, but these studies have analyzed the complex mixtures ofanthocyanins naturally occurring in the juices and extracts. Howchanging pH affects the color characteristics of individual anthocyaninmolecules or fractions of anthocyanins separated from natural sources,however, is not well understood or predictable. The prior art disclosesthat the number and types of substituents, e.g., the sugar and acylgroups, impact color; however, it does not disclose and it is not knownhow these substituents affect color as pH changes. Finally, although theprior art hypothesizes that various inter- and intra-molecularinteractions affect color, it does not disclose how changing pH affectsthese inter- and intra-molecular interactions and, ultimately, theobserved color of the anthocyanins.

WO 2009/100165 A2 discloses a method of separating anthocyanins fromother phenolic molecules in the juice of anthocyanin-containing fruitsand vegetables. WO 2009/100165 A2 does not disclose selectivelyseparating fractions of anthocyanin molecules based on differences incharge and polarity of the molecules to produce fractions with a desiredcolor that is different than the anthocyanin-containing juice.

The separation of individual anthocyanins at analytical scale isdescribed in J. Chromatography A., 1148 (2007), 38-45. The separation isconducted at low pH, i.e., pH of less than 2, using HPLC in order toassist in identifying individual anthocyanins. This method separatesanthocyanin molecules for detection rather than producing fractions withmixtures of anthocyanins.

WO 2004/012526 discloses a blue colorant solution of red cabbageanthocyanins at a pH of 7.9 that is used in a sugar-syrup for coatingconfectionery cores. The red cabbage anthocyanins were not separatedinto fractions.

There is no example in the prior art of isolating fractions ofanthocyanin molecules from anthocyanin-containing vegetable and fruitjuices and extracts at a select pH based on differences in charge andpolarity of the anthocyanin molecules. In addition, methods forobtaining anthocyanin fractions that provide different colorcharacteristics than those provided by the source juices and extractshave not been disclosed. In particular, the prior art has not describeda method for obtaining a natural blue anthocyanin-containing colorantcomposition providing color characteristics similar to those provided bythe synthetic blue colorant, FD&C Blue No. 1.

It is desirable to have a broad palette of natural colorants availablefor coloring foods. There is a long-felt need for natural blue colorantsthat provide color characteristics similar to those provided bysynthetic FD&C Blue No. 1. Therefore, a method of obtaining such naturalblue colorants from anthocyanin-containing vegetable and fruit juicesand extracts is desired.

SUMMARY OF THE INVENTION

The present invention is directed to a method of obtaining natural blueanthocyanin-containing colorant compositions providing colorcharacteristics similar to those provided by the synthetic bluecolorant, FD&C Blue No. 1. The natural blue anthocyanin-containingcolorant is obtained from anthocyanin-containing vegetable and fruitjuices and extracts by isolating fractions containing a mixture ofanthocyanin molecules at a select pH based on differences in charge andpolarity of the anthocyanin molecules.

In an embodiment, the invention is directed to a method of isolating afraction of anthocyanins from an anthocyanin-containing vegetable orfruit juice or extract, or a combination thereof comprising: a) loadingan anthocyanin-containing vegetable or fruit juice or extract, or acombination thereof, on an ion exchange column; b) selectivelyseparating anthocyanins on the ion exchange column based on differencesin charge and polarity of the anthocyanin molecules using a solvent ofselect pH, wherein the pH value is at least about 2; and c) selectingone fraction or a combination of fractions containing separatedanthocyanins, such that the separated anthocyanins in the one fractionor the combination of fractions, when in an aqueous solution at pH 8.0has a maximum absorbance of 615 nm to 635 nm. The selectedanthocyanin-containing fraction or combination of fractions containseparated anthocyanins that provide color characteristics closer tothose provided by FD&C Blue No. 1, but are different, i.e., afractionated subset, from that of the anthocyanin-containing vegetableor fruit juice or extract, or the combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two perspectives of a three dimensional representation ofthe color characteristics provided by FD&C Blue No. 1 in CIE 1976 CIELABL*a*b* color space as a function of concentration in aqueous solution.

FIG. 2 shows two perspectives of a three dimensional representation ofthe color characteristics provided by FD&C Blue No. 1 in CIE 1976 CIELCHL*C*h° color space as a function of concentration in aqueous solution.

FIG. 3 represents two perspectives of the area in CIE 1976 CIELAB L*a*b*color space of colors that differ from the colors provided by FD&C BlueNo. 1 by a ΔE of 3 or less and an illustration of a segmented tubedefined by the color space data.

FIG. 4 shows a comparison of the colors provided by different fruit andvegetable extracts in aqueous solution at different pH values.

FIG. 5 shows two perspectives of a three dimensional representation ofthe color characteristics provided by FD&C Blue No. 1 in CIE 1976 CIELABL*a*b* color space as a function of concentration in aqueous solution aswell as the area of colors that differ from the colors provided by BlueNo. 1 by a ΔE of 3 or less and also shows two perspectives of a threedimensional representation of the color characteristics provided bySpirulina Blue as a function of concentration in aqueous solution (whiteline closer to the x-axis).

FIG. 6 shows HPLC chromatograms at 520 nm detection of red cabbageextract solution and two fractions isolated from red cabbage extractsolution using a strong cation exchange column.

FIG. 7 shows HPLC chromatograms at 520 nm detection of red cabbageextract solution and four fractions isolated from red cabbage extractsolution using a strong cation exchange column.

FIG. 8 shows HPLC chromatograms at 520 nm detection of red cabbageextract solution identifying two groups of peaks that were targeted forisolating. These two groups of peaks were isolated as the “520-nmFraction” and the “530-nm Fraction.”

FIG. 9 provides a visual comparison of the colors provided by the 520-nmand 530-nm Fractions at different pH values. FIG. 9 also allows for avisual comparison of the colors provided by the 520-nm and 530-nmFractions with the color of a confectionery product panned with asugar-syrup colored with FD&C Blue No. 1.

FIG. 10 shows HPLC chromatograms at 520 nm detection of red cabbageextract solution and two fractions isolated from red cabbage extractsolution using semi-preparative HPLC. FIG. 10 shows that the 520-nm and530-nm Fractions each contain three distinct anthocyanin compounds andidentifies the functional groups and sugars on the anthocyanincompounds.

DETAILED DESCRIPTION OF THE INVENTION

Anthocyanin-containing vegetable and fruit juices and extracts arepresently used as natural, edible colorants and to produce colorantcompositions, in particular, natural red, purple, and blue hue colorantcompositions. The juices and extracts contain a mixture of all theanthocyanin molecules naturally present in the vegetable and fruitsources, along with numerous other classes of compounds. Therefore, thepresently available anthocyanin colorants are limited to those colorsassociated with the mixtures of anthocyanins that naturally exist in thevegetable and fruit sources. The invention involves methods of isolatingmixtures of anthocyanin molecules different from the complex mixture ofanthocyanins naturally present in vegetable and fruit juices andextracts. The method involves isolating fractions of anthocyaninmolecules from the complex mixture in the vegetable and fruit juices andextracts at a select pH based on differences in the charge and polarityof the anthocyanin molecules.

One aspect of the invention involves isolating fractions of anthocyaninmolecules from anthocyanin-containing vegetable and fruit juices andextracts to obtain colorant compositions providing specific, targetedcolor characteristics similar to those provided by the synthetic bluecolorant, FD&C Blue No. 1. As used herein, providing colorcharacteristics “similar” to FD&C Blue No. 1 means the color is closerin color characteristics than any other natural colorant, such as forexample, Spirulina Blue.

The applicants discovered that separating anthocyanins using a solventat a select pH and differences in polarity of the anthocyanin moleculeswould yield fractions containing mixtures of anthocyanins providingcolor characteristics similar to those provided by the synthetic bluecolorant, FD&C Blue No. 1. Each anthocyanin source contains differentamounts of multiple, distinct anthocyanin molecules, and each moleculemay exist in equilibrium with one or more secondary structures. Theremay be differences in charge and/or polarity among the differentanthocyanin molecules and their equilibrium molecular structures.Through separation based on differences in charge and polarity of theanthocyanin molecules at a select pH, the applicants were able toisolate fractions of anthocyanins with distinct spectral characteristicsfrom a complex mixture of anthocyanins. The spectral characteristics ofthe fractions were different and not evident from the spectralcharacteristics of the complex mixture of anthocyanins found in thejuice or extract. The applicants have identified anthocyanin fractionsthat provide color characteristics closer to those provided by syntheticFD&C Blue No. 1 than any known natural blue colorant can provide,including Spirulina Blue.

An “anthocyanin-containing vegetable or fruit juice” may be obtained bypressing liquid out of the fruit or vegetable. An“anthocyanin-containing vegetable or fruit extract” may be obtained bywashing a macerated fruit or vegetable with a solvent (e.g., water,alcohol). Juices and extracts contain anthocyanins as well as many othernaturally occurring compounds, including, for example, carbohydrates,acids, flavonoids, metal ions, phenolic acids, phenolic acid esters, andvitamins. The term, “vegetable or fruit juice or extract,” is equivalentto the list of terms, “vegetable juice, fruit juice, vegetable extract,or fruit extract,” and includes processed juices and extracts,including, for example, reconstituted juices and extracts, deodorizedjuices and extracts, and juices and extracts subjected to otherprocesses for removing specific or broad classes of compounds.

“Fractionation” is the process of selecting and separating a portion ofanthocyanins from the complex mixture of anthocyanins in ananthocyanin-containing vegetable or fruit juice or extract. The sourceof anthocyanins used in the method of the invention is ananthocyanin-containing vegetable or fruit juice or extract that providesblue hues at high pH values. In some embodiments, the source ofanthocyanins used in the method of the invention is red cabbage, purplesweet potato, blue potato, purple carrot or black carrot, or acombination thereof.

A “fraction” is the product of fractionation. An “anthocyanin fraction”contains a mixture of anthocyanins that is different from the mixture ofanthocyanins in the anthocyanin-containing juice or extract from whichthe fraction was separated. Anthocyanin fractions are separated from thejuice or extract at a select pH based on differences in charge andpolarity of the different anthocyanin molecules present.

A “select pH” is a pH of 2 or higher, e.g. a pH in a range of about 2 toabout 9, both in the context of separating and performing colorcharacterization of anthocyanins. In other embodiments the pH may be ata pH of 3 or higher, 4 or higher, 5 or higher, 6 or higher, or 7 orhigher, e.g., a pH in one of the following respective ranges, i.e.,about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 toabout 9 or about 7 to about 9.

“Maximum absorbance,” “lambda max,” or “λ_(max),” is the wavelength innanometers at which the maximum fraction of light is absorbed by asubstance. In general, the maximum absorbance can be used as acharacteristic value to compare substances when measured with aUV/Visible spectrophotometer or colorimeter.

References to “FD&C Blue No. 1” include the different names given to theidentical synthetic blue colorant, Brilliant Blue FCF and EuropeanCommission E133. The lambda max of FD&C Blue No. 1 is 630 nm.

A “colorant” is any substance that imparts color by absorbing orscattering light at different wavelengths. A “natural colorant” is acolorant that exists in or is produced by nature or is sourcedtherefrom. A “blue colorant” is a colorant that reflects light atwavelengths in the region of 450-495 nanometers and has a maximum UV/VISwavelength absorbance ranging from 615 to 635 nanometers. A “naturalanthocyanin-containing colorant” is a natural colorant comprisinganthocyanins sourced from plants.

The natural anthocyanin-containing colorant is a composition that maycomprise only anthocyanins or may also include other plant components.The composition may take the form of a solid, e.g., a powder, or aliquid solution, e.g., an aqueous liquid.

In an embodiment, the invention is directed to a method of isolating afraction of anthocyanins from an anthocyanin-containing vegetable orfruit juice or extract, or a combination thereof, comprising: a) loadingan anthocyanin-containing vegetable or fruit juice or extract, or acombination thereof, on an ion exchange column; b) selectivelyseparating anthocyanins on the ion exchange column based on differencesin charge and polarity of the anthocyanin molecules using a solvent of aselect pH; and c) selecting one fraction or a combination of fractionscontaining separated anthocyanins, such that the separated anthocyaninsin the one fraction or the combination of fractions, when in an aqueoussolution at pH 8.0 has a maximum absorbance of 615 nm to 635 nm. In anembodiment, the anthocyanin-containing fraction is separated from theanthocyanin-containing vegetable or fruit juice or extract with asolvent at a pH from a range of about 2 to about 9, or in one of thefollowing ranges, i.e., about 3 to about 9, about 4 to about 9, about 5to about 9, about 6 to about 9 or about 7 to about 9.

“Hue” refers to the color property that gives a color its name, forexample red, orange-red, blue, violet, etc.

“Chroma” is a color property indicating the purity of a color, wherehigher chroma is associated with greater purity of hue and less dilutionby white, gray, or black.

“Value” is a color property indicating the lightness or darkness of acolor, where higher value is associated with greater lightness.

The terms “color” and “color characteristics” are used interchangeably,and encompass color properties such as hue, chroma, and value and colormodel system parameters used to describe these properties, such asCommission Internationale de l'Eclairage CIE 1976 CIELAB color spaceL*a*b* values and CIELCH color space L*C*h° values. The CIELAB andCIELCH color models provide more perceptually uniform color spaces thanearlier color models. Colorants are analyzed with a spectrophotometer,and CIELAB L*a*b* and CIELCH L*C*h° values are calculated from thespectral data. The L*a*b* and L*C*h° values provide a means ofrepresenting color characteristics and assessing the magnitude ofdifference between two colors. The CIELAB L*a*b* and CIELCH L*C*h°values presented herein, in all instances unless stated otherwise, werecalculated from spectral data obtained from a Konica MinoltaSpectrophotometer CM-3500d operated in transmittance mode, with CIEStandard Illuminant D65 and 10 degree observer angle.

L*a*b* values consist of a set of coordinate values defined in athree-dimensional Cartesian coordinate system. L* is the value, orlightness, coordinate. L* provides a scale of lightness from black (0 L*units) to white (100 L* units) on a vertical axis. a* and b* arecoordinates related to both hue and chroma. a* provides a scale forgreenness (− a* units) to redness (+a* units), with neutral at thecenter point (0 a* units), on a horizontal axis. b* provides a scale forblueness (− b* units) to yellowness (+b* units), with neutral at thecenter point (0 b* units), on a second horizontal axis perpendicular tothe first horizontal axis. The three axes cross where L* has a value of50 and a* and b* are both zero.

L*C*h° values consist of a set of coordinate values defined in athree-dimensional cylindrical coordinate system. L* is the value, orlightness, coordinate. L* provides a scale of lightness from black (0 L*units) to white (100 L* units) on a longitudinal axis. h° is the huecoordinate. h° is specified as an angle from 00 to 3600 movingcounterclockwise around the L* axis. Pure red has a hue angle of 00,pure yellow has a hue angle of 90°, pure green has a hue angle of 180°,and pure blue has a hue angle of 270°. The C* coordinate representschroma and is specified as a radial distance from the L* axis. C*provides a scale from achromatic, i.e., neutral white, gray, or black,at the L* axis (0 C* units) to greater purity of hue as the coordinatemoves away from the L* axis (up to 100 or more C* units). C* and h° canbe calculated from a* and b* using Equations 1 and 2:

$\begin{matrix}{C^{*} = \left( {a^{*^{2}} + b^{*^{2}}} \right)^{0.5}} & (1) \\{{h\;{^\circ}} = {\arctan\mspace{11mu}\left( \frac{b^{*}}{a^{*}} \right)}} & (2)\end{matrix}$

“Delta E,” “ΔE_(ab)*,” or “ΔE” is a measure of the magnitude of totalcolor difference between two colors represented in CIELAB L*a*b* colorspace. It has been reported that an experienced color observer cannotdistinguish any difference between two colors when the ΔE is about 2.3or less. The ΔE of two different colors with L*a*b* values, L*₁a*₁b*₁and L*₂a*₂b*₂, is calculated using Equation 3:ΔE _(ab)*=√{square root over ((L* ₁ −L* ₂)²+(a* ₁ −a* ₂)²+(b* ₁ −b*₂)²)}  (3)

The CIELAB L*a*b* and CIELCH L*C*h° values of FD&C Blue No. 1 at sevendifferent concentrations in aqueous solution are presented in Table 1.These values were calculated from spectral data obtained with a KonicaMinolta Spectrophotometer CM-3500d using the transmittance setting.

TABLE 1 Concentration L* a* b* C* h° 1000 ppm  10.49 15.82 −44.99 47.69289.37 500 ppm 24.07 9.80 −58.18 59.00 279.56 100 ppm 52.43 −29.57−57.38 64.55 242.74  50 ppm 63.64 −43.71 −48.31 65.14 227.86  10 ppm84.25 −37.23 −23.42 43.99 212.17  5 ppm 90.65 −24.40 −14.28 28.27 210.33 1 ppm 97.69 −6.43 −3.57 7.36 209.02 These L*a*b* and L*C*h° values forFD&C Blue No. 1 represent the ideal target values for a natural bluecolorant alternative to FD&C Blue No. 1. Natural blue colorants havingL*a*b* values that fall within a ΔE of 2.3 or less from these targetvalues would be expected to provide color characteristics sufficientlysimilar to those provided by FD&C Blue No. 1 that a human eye could notdistinguish the difference in color provided by the natural colorantversus the synthetic. Clearly, the closer the L*a*b* values for anatural blue colorant come to the synthetic target values (i.e.,yielding smaller values of ΔE), the better replacement the natural bluecolorant will be for FD&C Blue No. 1 in an edible application.

FIG. 1 shows two perspectives of a three dimensional representation ofthe L*a*b* values for aqueous solutions of FD&C Blue No. 1 at the sevenconcentrations reported in Table 1, connected by line segments. FIG. 2shows two perspectives of a three dimensional representation of theL*C*h° values for aqueous solutions of FD&C Blue No. 1 at the sevenconcentrations reported in Table 1, connected by line segments.

Mathematical models can be generated to represent the colorcharacteristics provided by FD&C Blue No. 1 at any concentration in theL*a*b* and L*C*h* color spaces. For example, the color characteristicsmay be represented by a segmented line model connecting the L*a*b* orL*C*h° data points of Table 1. A line (L) connecting two points (P¹ andP²) representing two different concentrations of FD&C Blue No. 1 inL*a*b* space can be calculated with the following Equation 4:L={P ₁ +t*(P ₂ −P ₁)}  (4)

-   -   wherein P₁ is (L*₁, a*₁, b*₁); P₂ is (L*₂, a*₂, b*₂); and t is        any real number.

Consequently, a segmented line model for FD&C Blue No. 1 in L*a*b* colorspace can be interpolated based on the L*a*b* values for the sevendifferent concentration points using Equation 4 as follows:

-   -   For concentrations between 500 and 1000 ppm, 0<t<1:        -   L*=10.49+13.58*t        -   a*=15.82+−6.02*t        -   b*=−44.99+−13.19*t    -   For concentrations between 100 and 500 ppm, 0<t<1:    -   L*=24.07+28.36*t    -   a*=9.80+−39.37*t    -   b*=−58.18+0.80*t    -   For concentrations between 50 and 100 ppm, 0<t<1:        -   L*=52.43+11.21*t        -   a*=−29.57+−14.14*t        -   b*=−57.38+9.07*t    -   For concentrations between 10 and 50 ppm, 0<t<1:        -   L*=63.64+20.61*t        -   a*=−43.71+6.48*t        -   b*=−48.31+24.89*t    -   For concentrations between 5 and 10 ppm, 0<t<1:        -   L*=84.25+6.40*t        -   a*=−37.23+12.83*t        -   b*=−23.42+9.14*t    -   For concentrations between 1 and 5 ppm, 0<t<1:        -   L*=90.65+7.04*t        -   a*=−24.40+17.97*t        -   b*=−14.28+10.71*t            The segmented line model for FD&C Blue No. 1 in L*a*b* space            is drawn in FIG. 1.

In addition, colors having L*a*b* values falling within a specific ΔErange of the FD&C Blue No. 1 model can be mathematically modeled inL*a*b* color space. Selecting a specific ΔE value, e.g., 3, with respectto FD&C Blue No. 1 and plotting that ΔE in L*a*b* color space results ina tube-like structure around the FD&C Blue No. 1 model, as shown in FIG.3. It is noted that any color with a ΔE value of about 2.3 or less fromany point on the model will not be distinguishable from the colorprovided by FD&C Blue No. 1.

To determine whether a point (X₀) in L*a*b* color space falls within aspecific ΔE value from the FD&C Blue No. 1 model, the minimum distance,d_(min), between the point and the model (represented by line segment X₁to X₂) must be calculated.

Equation 5 can be used to calculate d_(min):

$\begin{matrix}{d_{\min} = \frac{{\left( {x_{0} - x_{1}} \right) \times \left( {x_{0} - x_{2}} \right)}}{{x_{2} - x_{1}}}} & (5)\end{matrix}$

-   -   wherein x denotes the cross product of two vectors and vertical        bars denote the magnitude of a vector expression.        If the value of d_(min) is less than or equal to the chosen ΔE        value, then the point in L*a*b* color space falls within that        specific ΔE value from the FD&C Blue No. 1 model.

For example, it may be determined whether Spirulina Blue provides acolor having a ΔE of 12 or less compared to the color provided by FD&CBlue No. 1. Table 2 shows the color characteristics provided bySpirulina Blue, a known natural blue colorant, at two differentconcentrations in aqueous solution:

TABLE 2 Concentration L* a* b* C* h° (404.8 mg/L) 69.97 −29.69 −43.5652.72 253.72  (206 mg/L) 80.3 −23.97 −29.39 37.92 230.8The X₀ for the 404.8 mg/L Spirulina Blue solution in L*a*b* color spaceis:

-   -   X₀=(69.97, −29.69, −43.56)        The X₀ for the 206 mg/L Spirulina Blue solution in L*a*b* color        space is:    -   X₀=(80.3, −23.97, −29.39)        X₁ and X₂ are two points from the FD&C Blue No. 1 model at 10        ppm and 50 ppm concentration in an aqueous solution,        respectively:    -   X₁=(63.64, −43.71, −48.31)    -   X₂=(84.25, −37.23, −23.24)

The d_(min), calculated using Equation 5, is 12.4 for the 404.8 mg/LSpirulina Blue solution and 14.4 for the 206 mg/L Spirulina Bluesolution. Therefore, the Spirulina Blue solutions do not provide a colorhaving a ΔE of 12 or less compared to the color provided by FD&C BlueNo. 1 in aqueous solution when measured against the segmented linedefined by the L*a*b* values for 10 ppm and 50 ppm FD&C Blue No. 1 inaqueous solution.

Spectral characteristics of a number of different solutions of SpirulinaBlue were determined as shown in Table 3.

TABLE 3 Spirulina Solutions Data Data Name ppm L*(D65) a*(D65) b*(D65)C*(D65) h°(D65) 0.04% Spirulina 400 67.69 −30.25 −45.87 54.94 236.60.03% Spirulina 300 72.77 −29.43 −39.52 49.27 233.32 0.02% Spirulina 20078.87 −25.56 −30.99 40.17 230.49 0.015% Spirulina  150 82.98 −21.82−25.29 33.4 229.22 0.01% Spirulina 100 87.77 −16.29 −18.32 24.52 228.350.0075% Spirulina  75 90.46 −12.94 −14.27 19.27 227.79 0.005% Spirulina 50 93.23 −9.26 −10.13 13.72 227.59

-   -   The data for Blue Spirulina has been plotted in the color graphs        shown in FIG. 5 versus the FD&C Blue No. 1 data.

Differences between the color characteristics provided by Spirulina Blueand FD&C Blue No. 1 are represented in FIG. 5. FIG. 5 shows thesegmented line model of the color characteristics provided by FD&C BlueNo. 1 in L*a*b* color space at concentrations from 1 ppm to 1000 ppm inaqueous solution, with the model surrounded by a tube representing thearea of colors that differ from the colors provided by Blue No. 1 by aΔE of 3 or less. For comparison, FIG. 5 also shows a segmented linemodel of the color characteristics provided by Spirulina Blue in L*a*b*color space at concentrations from 50 ppm to 400 ppm in aqueoussolution. The Spirulina Blue model does not intersect the Blue No. 1model or associated tube at any point in L*a*b* color space.

The invention includes selecting a fraction or combination of fractionshaving natural blue anthocyanin-containing colorants sourced fromvegetable, fruit or combinations thereof. The fraction or combination offractions comprise a selectively separated mixture of anthocyanins,wherein at least one concentration of the colorant when in an aqueoussolution at pH 8.0 provides color characteristics having a ΔE value of12 or less compared to the color characteristics defined by thesegmented line defined by the L*a*b* values of 5 ppm and 10 ppm FD&CBlue No. 1 in aqueous solution. In other embodiments the ΔE value may beless than 11, 10, 9, 8, 7, 6, 5, 4 or 3. The at least one concentrationof colorant may also if desired be measured against a plurality ofsegmented lines defined by different concentrations of FD&C Blue No. 1in aqueous solution, e.g., 1 and 5 ppm, 10 ppm and 50 ppm, 50 ppm and100 ppm, 100 ppm and 500 ppm, 500 ppm and 1000 ppm, or any combinationselected therefrom. For example, while not required, the at least oneconcentration of a colorant may be defined as having a ΔE value of 12 orless for a first segmented line at 5 ppm to 10 ppm, a ΔE value of 8 orless for a segmented line at 1 to 5 ppm and ΔE value of 12 or less for asegmented line at 10 ppm to 50 ppm. However, if ΔE value is used todescribe the colorant of the invention, only one segmented line isrequired to define the colorant.

While Spirulina Blue is the natural colorant considered to provide theclosest color match to FD&C Blue No. 1, the natural blueanthocyanin-containing colorant sourced from vegetable, fruit orcombinations thereof that is a selectively separated mixture ofanthocyanins in a fraction or combination of fractions obtained inaccordance with the method of this invention is a better color match. Inparticular, when at least one concentration of the colorant in theselected fraction or combination of fractions is in an aqueous solutionat pH 8.0, that colorant aqueous solution provides color characteristicsmatching a FD&C Blue No. 1 segmented line based on a series of aqueoussolutions having differing concentrations of FD&C Blue No. 1 defined inan L*a*b* color space, wherein matching means the at least oneconcentration of the colorant in an aqueous solution at pH of 8.0 has aΔE value measured against the FD&C Blue No. 1 segmented line that is atleast one unit less than a ΔE value for a Spirulina Blue segmented linedefined in the same L*a*b* color space based on a series of aqueoussolutions having differing concentrations of Spirulina Blue measuredagainst FD&C Blue No. 1 segmented line. In other embodiments the ΔEvalue of the at least one concentration of the colorant in an aqueoussolution at pH of 8.0 measured against the FD&C Blue No. 1 segmentedline is at least 2, 3, 4, 5 or 6 units less than a ΔE value for aSpirulina Blue segmented line measured against FD&C Blue No. 1 segmentedline. In still other embodiments the ΔE value of the at least oneconcentration of the colorant in an aqueous solution at pH of 8.0measured against the FD&C Blue No. 1 segmented line is at least 7, 8, 9,10 or 11 units less than a ΔE value for a Spirulina Blue segmented linemeasured against FD&C Blue No. 1 segmented line.

Various fruit and vegetable extracts containing anthocyanins wereanalyzed to identify a source of anthocyanins that would provide colorcharacteristics closest to those provided by the synthetic bluecolorant, FD&C Blue No. 1. FIG. 4 shows a comparison of six differentcommercially available extracts of red cabbage, purple sweet potato,black carrot, red radish, purple corn, and grape in aqueous solution atfive different pH values. Visually, it can be seen that anthocyaninsfrom red radish, purple corn, and grape did not provide blue hues inaqueous solution at any pH in the range from pH 6 to pH 8. Anthocyaninsfrom red cabbage, purple sweet potato, and black carrot provided bluehues in aqueous solution at the higher end of the pH range.

Any anthocyanin-containing fruit or vegetable juice or extract thatprovides blue hues at high pH values may be used as a source ofanthocyanins to produce anthocyanin fraction(s) of the invention. Insome embodiments, the anthocyanin fraction is isolated from an extractof red cabbage, purple sweet potato, blue potato, purple carrot or blackcarrot, or a combination thereof.

In an embodiment, the method involves selectively isolatinganthocyanin-containing fractions from red cabbage extract to produce anatural anthocyanin-containing colorant providing color characteristicssimilar to those provided by synthetic FD&C Blue No. 1.

Selected anthocyanin-containing fractions of anthocyanin-containingfruit and vegetable juices and extracts may be isolated using an ionexchange column or semi-preparative HPLC column. Suitable ion exchangemedia include cation and anion exchange media. Suitable semi-preparativeHPLC columns include C-18 columns. In an embodiment, the ion exchangecolumn is activated with a solvent appropriate to the ion exchange mediaprior to loading of a vegetable or fruit juice or extract.

The anthocyanin-containing fraction is separated from theanthocyanin-containing vegetable or fruit juice or extract with asolvent at a pH of at least about 2, preferably at least about 4. Insome embodiments, the anthocyanin fraction is separated with a solventat a pH from about 2 to about 9. In yet another embodiment, theanthocyanin fraction is separated with a solvent at a pH from about 3 toabout 9. In yet another embodiment, the anthocyanin fraction isseparated with a solvent at a pH from about 4 to about 9. In yet anotherembodiment, the anthocyanin fraction is separated with a solvent at a pHfrom about 5 to about 9. In other embodiments, the anthocyanin fractionis separated with a solvent at a pH from about 6 to about 9. In stillother embodiments, the anthocyanin fraction is separated with a solventat a pH from about 7 to about 9.

Suitable solvents for eluting the selected anthocyanin-containingfractions include methanol, acetonitrile, water, and mixtures thereof,depending on the polarity of the column media and the solubility of theanthocyanin-containing juice or extract. In some embodiments, thesolvent is an aqueous methanol solution.

Suitable agents that may be added to the solvent to adjust pH includepotassium phosphate, sodium hydroxide, and the like.

In yet another embodiment, the invention is directed to a method ofisolating a second fraction of anthocyanins from theanthocyanin-containing vegetable or fruit juice or extract, or acombination thereof, comprising: a) selectively separating anthocyaninson the ion exchange column based on differences in charge and polarityof the anthocyanin molecules using a second solvent of a select pH,wherein the pH value of the second solvent is different from, preferablyhigher than, the pH value of the solvent used to elute the firstfraction; and b) selecting a second fraction or combination of fractionscontaining separated anthocyanins, such separated anthocyanins in thesecond fraction or the combination of fractions, when in an aqueoussolution at pH of 8.0 provides color characteristics of those providedby FD&C Blue No. 1 as measured by having a maximum absorbance of 615 nmto 635 nm. In this embodiment, the first fraction, which may beseparated with a first solvent of select pH, such as a solvent of pH ofat least about 2, from the ion exchange column does not provide amixture of separated anthocyanins that when in an aqueous solution at pHof 8.0 provides color characteristics of those provided by FD&C Blue No.1 as measured by having a maximum absorbance of 615 nm to 635 nm. In anembodiment, the selected second anthocyanin-containing fraction isseparated from the anthocyanin-containing vegetable or fruit juice orextract with a solvent at a pH from about 2 to about 9, or in one of thefollowing ranges of increasing preference, i.e., about 3 to about 9,about 4 to about 9, about 5 to about 9, about 6 to about 9 or mostpreferably about 7 to about 9.

Additional anthocyanin-containing fractions may be isolated by furtherfractionating a selected anthocyanin-containing fraction using an ionexchange column or semi-preparative HPLC column. Suitable ion exchangemedia include cation and anion exchange media. Suitable semi-preparativeHPLC columns include C-18 columns.

For example, in another embodiment, the fractionation method may furthercomprise the steps of: c) loading the selected one fraction orcombination of fractions containing separated anthocyanins on an ionexchange column; d) selectively separating the anthocyanins loaded onthe ion exchange column in step c) based on differences in charge andpolarity of the anthocyanin molecules using a solvent of select pH; ande) selecting one fraction or a combination of fractions containingseparated anthocyanins separated in step d) such that the separatedanthocyanins selected in step e) when in an aqueous solution at a pH of8.0 provides a maximum absorbance of 620 nm to 635 nm. Third, fourth andfurther additional anthocyanin-containing fractions may be produced in asimilar manner if desired. In yet another embodiment, the separatedanthocyanins selected in step e) in at least one concentration in anaqueous solution at pH of 8.0 provides color characteristics having a ΔEvalue of 12 or less compared to the color characteristics defined by thesegmented line defined by the L*a*b* values of 5 ppm and 10 ppm FD&CBlue No. 1 in aqueous solution.

In yet another embodiment, the step of selectively separatinganthocyanins on the ion exchange column based on differences in chargeand polarity of the anthocyanin molecules comprises the steps of (i)first using the solvent of select pH to obtain a first fraction and (ii)using a second solvent of second select pH, wherein the pH value of thesecond solvent is different from the pH value of the first solvent toobtain a subsequent fraction that is the one fraction or to obtain acombination of a plurality of subsequent fractions that is thecombination of fractions, wherein the separated anthocyanins in the onefraction or combination of fractions when in at least one concentrationin an aqueous solution at pH of 8.0 provides color characteristicshaving a ΔE value of 12 or less compared to the color characteristicsdefined by the segmented line defined by the L*a*b* values of 5 ppm and10 ppm FD&C Blue No. 1 in aqueous solution.

In yet another embodiment, the step of selectively separatinganthocyanins on the ion exchange column based on differences in chargeand polarity of the anthocyanin molecules comprises the steps of (i)first using the solvent of select pH which is a first eluting solvent toobtain a first fraction and (ii) using one or more subsequent elutingsolvents of select pH to obtain the one fraction or combination offractions, wherein each eluting solvent is different, and the differencemay be independently selected from the group of pH, solvent make-up anda combination thereof. Preferably the select pH of the first elutingsolvent is lower than the select pH of the one or more subsequentfractions. Preferably the select pH will range from about 2 to about 9,or in one of the following ranges of increasing preference, i.e., about3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9or most preferably about 7 to about 9. In yet another embodiment, theseparated anthocyanins in the one fraction or the combination offractions, when at least one concentration is in an aqueous solution atpH of 8.0, provides color characteristics having a ΔE value of 12 orless compared to the color characteristics defined by the segmented linedefined by the L*a*b* values of 5 ppm and 10 ppm FD&C Blue No. 1 inaqueous solution.

Isolated anthocyanin fractions may be used as colorants, or may befurther processed by, for example, purification, concentration,deodorization, or color stabilization.

The selective separation method can be performed at a scale thatproduces commercially useful quantities of natural blue colorants.

The natural blue anthocyanin-containing colorants prepared by the methodof this invention may be applied to or incorporated into all types ofedible products, including foods for human and animal consumption,beverages, and pharmaceutical products. Examples of edible productsinclude pet food and treats, dry goods (e.g., rice, grains, andcereals), soups and sauces, confectionery products (e.g., chocolates,sugar and sugarless candies of all types, chewing gum, candy bars, andsugar-coated confectionery), dessert products (e.g., pudding, frosting,icing, and toppings), baked goods (e.g., cakes, cookies, wafers, andbiscuits), dairy products (e.g., yogurt, whipped cream, and cheese),beverages (e.g., dairy-based drinks, waters, juices, teas, and sodas),snack products (e.g., crackers, snack bars, pretzels, and chips), andpharmaceutical forms (e.g., tablets, suspensions, chewables, andsyrups). The natural blue anthocyanin-containing colorant may also beincorporated into food grade colorant compositions, coatings, and inks.In an embodiment, the blue anthocyanin-containing colorant is includedin a coating or ink applied to a surface of a confectionery product. Inanother embodiment, the blue anthocyanin-containing colorant is includedin a coating or ink applied to a surface of a confectionery product,wherein the confectionery product is a confectionery center with a softpanned or hard panned sugar-based coating. In yet another embodiment,the blue anthocyanin-containing colorant is included in a coating or inkapplied to a surface of a confectionery product, wherein theconfectionery product is a confectionery center with a soft panned orhard panned sugarless coating.

In an embodiment, a red cabbage extract solution is fractionated using astrong cation exchange column. A first fraction is eluted with 75% v/v0.1 M potassium phosphate buffer at pH 8 and 25% v/v methanol. A secondfraction is eluted with 30% v/v 0.1 M potassium phosphate buffer at pH 8and 70% v/v methanol.

In another embodiment, a red cabbage extract solution is fractionatedusing a strong cation exchange column. A first fraction is eluted with75% v/v 0.1 M potassium phosphate buffer at pH 6 and 25% v/v methanol. Asecond fraction is eluted with 75% v/v 0.1 M potassium phosphate bufferat pH 7 and 25% v/v methanol. A third fraction is eluted with 75% v/v0.1 M potassium phosphate buffer at pH 8 and 25% v/v methanol. A fourthfraction is eluted with 30% v/v 0.1 M potassium phosphate buffer at pH 8and 70% v/v methanol.

In another embodiment, a red cabbage extract solution is separated usinga C-18 semi-preparative HPLC column.

The method of selectively separating anthocyanin fractions from complexmixtures of anthocyanins in vegetable and fruit juices and extractsbased on differences in charge and polarity of the anthocyanin moleculesyields colorants providing color characteristics that are different fromthose provided by the complex mixtures.

This method of selectively separating anthocyanin fractions from complexmixtures of anthocyanins based on differences in polarity of theanthocyanin molecules fulfills the long-felt need for a means ofobtaining natural colorants providing color characteristics similar tothose provided by the synthetic colorant, FD&C Blue No. 1.

Specific embodiments of the invention will now be demonstrated byreference to the following examples. It should be understood that theseexamples are disclosed solely by way of illustrating the invention andvariations within the spirit of the invention are anticipated.

Example 1

Fractionation of Red Cabbage Extract Using Strong Cation ExchangeCartridge

An SCX (Strong Cation Exchange) solid phase extraction cartridge fromPhenomenex® (Torrance, Calif.) was activated using pure methanol. Thecartridge was washed using 0.01% v/v acidified water. An aqueoussolution of red cabbage extract was loaded into the cartridge and washedwith 0.01% v/v acidified water. A potassium phosphate buffer (0.1 M) atpH 8 was passed through the cartridge. Fraction 1 was eluted andcollected using a 25% v/v methanol solution at pH 8. Fraction 2 waseluted and collected using a 70% v/v methanol solution at pH 8.

Fractions 1 and 2 were acidified with 2-5 ml of 88% v/v formic acid. Themethanol was removed using a rotary evaporator.

In order to remove any salts, Fraction 1 was loaded into a C-18cartridge and eluted with 0.01% v/v acidified water. The eluent wascollected in 0.01% v/v acidified water, and the residual methanol wasevaporated. Fraction 2 was also passed through a C-18 cartridge usingthe same procedure outlined for Fraction 1.

The maximum UV/VIS wavelength absorbance and color characteristicsprovided by the red cabbage extract solution (RCE) and Fractions 1 and 2were analyzed at different pH values as shown below in Table 4.

TABLE 4 pH λ_(max) L* a* b* C* h° 6.0 RCE 552.80 93.86 2.48 −2.73 3.69312.31 Fraction 1 551.40 94.43 2.24 −2.24 3.17 314.98 Fraction 2 553.6093.63 2.64 −3.29 4.22 308.79 6.6 RCE 560.80 92.86 1.74 −3.89 4.27 249.07Fraction 1 558.20 93.54 1.75 −3.31 3.75 297.89 Fraction 2 565.60 92.621.59 −4.46 4.73 289.62 7.0 RCE 596.80 92.65 −0.49 −4.10 4.13 263.14Fraction 1 594.0 92.43 −0.22 −4.60 4.61 267.28 Fraction 2 599.80 92.07−1.17 −5.11 5.24 257.10 7.6 RCE 612.0 92.10 −3.23 −4.62 5.64 235.00Fraction 1 608.40 91.41 −3.47 −5.80 6.76 239.08 Fraction 2 616.40 91.62−4.17 −5.68 7.05 233.67 8.0 RCE 612.40 91.17 −5.05 −5.77 7.67 228.82Fraction 1 610.60 90.90 −5.26 −6.40 8.29 230.59 Fraction 2 619.40 91.56−5.80 −5.81 8.21 225.04Fraction 2 at pH 7.6 and pH 8.0 provided max values closest to that ofsynthetic FD&C Blue No. 1 (λ_(max)=630 nm), i.e., λ_(max) values of616.40 and 619.40, respectively.

ΔE values may also be calculated to compare the color characteristicsprovided by Fraction 2 at pH 7.6 and pH 8.0 to those provided bysynthetic FD&C Blue No. 1. The ΔE values are equivalent to the minimumdistances between the Fraction 2 color points in L*a*b* color space andthe FD&C Blue No. 1 model. Therefore, Equation 5 is used to calculatethe d_(min), or ΔE, values from the following data:

The X₀ for Fraction 2 at pH 7.6 in L*a*b* color space is:

-   -   X₀=(91.62, −4.17, −5.68)

The X₀ for Fraction 2 at pH 8.0 in L*a*b* color space is:

-   -   X₀=(91.56, −5.80, −5.81)

X₁ and X₂ are two points from the FD&C Blue No. 1 model:

-   -   X₁=(90.65, −24.40, −14.28)    -   X₂=(97.69, −6.43, −3.57)

The calculated d_(min), or ΔE, values are 6.7 for Fraction 2 at pH 7.6,and 6.0 for Fraction 2 at pH 8.0.

FIG. 6 provides HPLC chromatograms at 520 nm of the red cabbage extractsolution (RCE) and Fractions 1 and 2. FIG. 6 shows that Fraction 2 has ahigher concentration of the later-eluting peaks from the red cabbageextract solution.

Example 2

Fractionation of Red Cabbage Extract Using Strong Cation ExchangeCartridge and Solvents of Different High pH Values

An SCX (Strong Cation Exchange) solid phase extraction cartridge fromPhenomenex® (Torrance, Calif.) was used. A red cabbage extract dilutedin 0.01% v/v acidified water (10-15 ml) was loaded into the cartridgeand washed with 0.01% v/v acidified water. A potassium phosphate buffer(0.1 M) at pH 6 was passed through the cartridge. Fraction 1 was elutedand collected using a 25% v/v methanol solution at pH 6. A potassiumphosphate buffer (0.1 M) at pH 7 was passed through the cartridge.Fraction 2 was eluted and collected using a 25% v/v methanol solution atpH 7. A potassium phosphate buffer (0.1 M) at pH 8 was passed throughthe cartridge. Fraction 3 was eluted and collected using a 25% v/vmethanol solution at pH 8. Fraction 4 was eluted and collected using a70% v/v methanol solution at pH 8.

Fractions 1 to 4 were acidified with 20% v/v formic acid. The methanolwas removed using a rotary evaporator.

In order to wash the salts, Fraction 1 was loaded into a C-18 cartridgeand eluted with 0.01% v/v acidified water. The eluent was collected in0.01% v/v acidified water, and the residual methanol was evaporated.Fractions 2 to 4 were also passed through a C-18 cartridge using thesame procedure outlined for Fraction 1.

The maximum UV/VIS wavelength absorbance and color characteristicsprovided by the red cabbage extract solution (RCE) and Fractions 1 to 4were analyzed at different pH values as shown below in Table 5.

TABLE 5 pH λ_(max) L* a* b* C* h° 6.0 RCE 553.0 93.08 3.10 −3.52 4.69311.40 Fraction 1 549.8 95.21 1.64 −1.33 2.11 320.92 Fraction 2 552.494.75 1.99 −1.96 2.79 315.53 Fraction 3 552.0 94.42 2.19 −2.25 3.13314.20 Fraction 4 554.2 92.41 3.49 −4.46 5.66 307.99 7.0 RCE 596.0 91.07−0.77 −5.72 5.77 262.31 Fraction 1 592.6 93.37 −0.12 −3.36 3.36 267.91Fraction 2 591.6 92.59 0.19 −4.35 4.36 272.54 Fraction 3 594.4 92.32−0.38 −4.62 4.63 265.34 Fraction 4 601.8 90.65 −1.96 −6.52 6.81 253.308.0 RCE 612.6 90.00 −6.20 −6.84 9.23 227.77 Fraction 1 606.6 91.05 −4.44−5.87 7.36 232.93 Fraction 2 608.8 90.28 −5.40 −7.14 8.95 232.86Fraction 3 611.6 90.21 −5.92 −7.16 9.29 230.42 Fraction 4 622.2 90.08−7.87 −7.20 10.67 222.43Fraction 4 at pH 8.0 provided λ_(max) value closest to that of syntheticFD&C Blue No. 1 (λ_(max)=630 nm), i.e., a λ_(max) value of 622.2.

A ΔE value may also be calculated to compare the color characteristicsprovided by Fraction 4 at pH 8.0 to those provided by synthetic FD&CBlue No. 1. The ΔE value is equivalent to the minimum distance betweenthe Fraction 4 color point in L*a*b* color space and the FD&C Blue No. 1model. Therefore, Equation 5 is used to calculate the d_(min), or ΔE,value from the following data:

The X₀ for Fraction 4 at pH 8.0 in L*a*b* color space is:

-   -   X₀=(90.08, −7.87, −7.20)

X₁ and X₂ are two points from the FD&C Blue No. 1 model:

-   -   X₁=(90.65, −24.40, −14.28)    -   X₂=(97.69, −6.43, −3.57)

The calculated d_(min), or ΔE, value is 6.7 for Fraction 4 at pH 8.0.

FIG. 7 provides the HPLC chromatograms at 520 nm detection of the redcabbage extract solution (RCE) and Fractions 1 to 4. FIG. 7 shows thatFraction 4 has a higher concentration of the later-eluting peaks fromthe red cabbage extract solution.

Example 3

Separation of Red Cabbage Extract Peak Groups Using Semi-PreparativeHPLC

Fractions associated with two specific groups of peaks, as shown in thechromatogram of FIG. 7, may be separated and collected from red cabbageextract solution using semi-preparative HPLC. The red cabbage extractsolution was loaded onto a C-18 semi-preparative HPLC cartridge and twofractions, the 520-nm Fraction (λ_(max)=524 nm) and the 530-nm Fraction(λ_(max)=532 nm), were eluted using an acidic acetonitrile and watergradient. The residual acetonitrile was evaporated from each fractionwith a rotary evaporator.

Color characterization was performed after adjusting the concentrationsof the fractions and mixing separate fraction aliquots with buffer toproduce five aliquots at pH 6, 6.6, 7, 7.6, and 8. The maximum UV/VISwavelength absorbance and color characteristics of the 520-nm and 530-nmFraction aliquots were analyzed, and the results are provided in Table6.

TABLE 6 Abs Fraction pH λ_(max) (Amu) L* a* b* C* h° 520-nm 1-2 5242.161 80.05 33.41 −6.17 33.97 349.54 (107.71 6.0 ND¹ ND 95.95 0.95 −0.721.19 322.87 mg/L) 6.6 ND  ND 95.64 0.80 −1.08 1.34 306.49 7.0 585.80 0.299.32 0.48 −2.56 2.60 280.71 7.6 602.00 0.389 92.56 −1.72 −4.70 5.01249.91 8.0 603.80 0.488 92.09 −3.15 −5.17 6.06 238.68 530-nm 1-2 5380.752 89.83 13.86 −5.80 15.02 337.31 (55.60 6.0 554.40 0.610 89.27 6.07−8.06 10.10 306.98 mg/L) 6.6 587.00 0.707 87.64 1.43 −9.96 10.06 278.147.0 599.60 0.848 86.88 −2.85 −10.96 11.33 255.45 7.6 621.80 1.156 87.67−5.44 −9.90 11.30 241.23 8.0 621.00 1.294 86.39 −11.79 −11.98 16.81225.45 ¹ND indicates that the absorbance spectra of the sample did notshow a maximum peak in the visible range.The 530-nm Fraction has a maximum absorbance of about 621 nm at pH 7.6and pH 8.0 and provides λ_(max) closest to that of synthetic FD&C BlueNo. 1 (λ_(max)=630 nm).

ΔE values may also be calculated to compare the color characteristicsprovided by the 530-nm Fraction at pH 7.6 and pH 8.0 to those providedby synthetic FD&C Blue No. 1. The ΔE values are equivalent to theminimum distances between the 530-nm Fraction color points in L*a*b*color space and the FD&C Blue No. 1 model. Therefore, Equation 5 is usedto calculate the d_(min), or ΔE, values from the following data:

The X₀ for the 530-nm Fraction at pH 7.6 in L*a*b* color space is:

-   -   X₀=(87.67, −5.44, −9.90)

The X₀ for the 530-nm Fraction at pH 8.0 in L*a*b* color space is:

-   -   X₀=(86.39, −11.79, −11.98)

X₁ and X₂ are two points from the FD&C Blue No. 1 model:

-   -   X₁=(84.25, −37.23, −23.42)    -   X₂=(90.65, −24.40, −14.28)

The calculated d_(min), or ΔE, values are 12.1 for the 530-nm Fractionat pH 7.6, and 9.9 for the 530-nm Fraction at pH 8.0.

FIG. 9 provides a visual comparison of the 520-nm and 530-nm Fractionsat different pH values. The concentration of the 520-nm Fraction is107.7 mg/L (Cyn-3-glu) and the concentration of the 530-nm Fraction is55.6 mg/L (Cyn-3-glu). At neutral and higher pH, it can be seen that the530-nm Fraction provides two to four times the chroma (as measured byC*) of the 520-nm Fraction at half the colorant concentration.

FIG. 10 provides the HPLC chromatograms at 520 nm detection of the redcabbage extract solution and the 520-nm and 530-nm Fractions. FIG. 10indicates that each fraction contains three distinct anthocyanincompounds.

Comparative Example

Several different concentrations of the red cabbage anthocyanin solutiondisclosed in the Examples of WO 2004/012526 were prepared at pH of 8.0.There was no fractionation conducted to separate and collect separatedanthocyanin-containing colorants. The maximum absorbance of theresulting solutions was 610 nm. The color was not considered anacceptable match for the color of FD&C Blue No. 1.

What is claimed:
 1. A method of isolating a fraction of anthocyaninsfrom an anthocyanin-containing vegetable or fruit juice or extract, or acombination thereof, comprising: a) loading an anthocyanin-containingvegetable or fruit juice or extract, or a combination thereof, on an ionexchange column; b) selectively separating anthocyanins into two or morefractions on the ion exchange column based on differences in charge andpolarity of the anthocyanin molecules using a solvent of select pH,wherein the pH is from 2 to less than 4; c) selecting one fraction or acombination of fractions containing separated anthocyanins, such thatthe separated anthocyanins in the one fraction or the combination offractions, when in an aqueous solution at pH 8.0 has a maximumabsorbance of 615 nm to 635 nm; and d) adjusting a pH of the onefraction or the combination of fractions to obtain a solution comprisingseparated anthocyanins having a maximum absorbance of 615 nm to 635 nm.2. The method of claim 1, wherein the separated anthocyanins in the onefraction or the combination of fractions in at least one concentrationin an aqueous solution at pH of 8.0 provides color characteristicshaving a ΔE value of 12 or less compared to the color characteristicsdefined by the segmented line defined by the L*a*b* values of 5 ppm and10 ppm FD&C Blue No. 1 in aqueous solution.
 3. The method of claim 1,wherein the source of the anthocyanin-containing vegetable or fruitjuice or extract is selected from the group consisting of red cabbage,purple sweet potato, blue potato, black carrot, purple carrot andcombinations thereof.
 4. The method of claim 3, wherein the source ofthe anthocyanin-containing vegetable or fruit juice or extract is redcabbage.
 5. The method of claim 1, wherein a firstanthocyanin-containing fraction is eluted with a 25% v/v methanolsolution at pH 8 and a subsequent anthocyanin-containing fraction thatis the one fraction or a plurality of subsequent fractions that is thecombination of fractions is eluted with a 70% v/v methanol solution atpH
 8. 6. The method of claim 5, further comprising a step of purifyingthe one fraction or the combination of fractions.
 7. The method of claim6, wherein the separated anthocyanins of the one fraction or thecombination of fractions in at least one concentration in an aqueoussolution at pH of 8.0 provides color characteristics having a ΔE valueof 12 or less compared to the color characteristics defined by thesegmented line defined by the L*a*b* values of 5 ppm and 10 ppm FD&CBlue No. 1 in aqueous solution.
 8. The method of claim 1, wherein theion exchange column is a cation exchange column.
 9. The method of claim1, wherein the step of selectively separating anthocyanins on the ionexchange column based on differences in charge and polarity of theanthocyanin molecules comprises the steps of (i) first using the solventof select pH to obtain a first fraction and (ii) using a second solventof a second select pH, wherein the pH value of the second solvent isdifferent from the pH value of the first solvent to obtain a subsequentfraction that is the one fraction or to obtain a combination of aplurality of subsequent fractions that is the combination of fractions,wherein the separated anthocyanins in the one fraction or combination offractions when in at least one concentration in an aqueous solution atpH of 8.0 provides color characteristics having a ΔE value of 12 or lesscompared to the color characteristics defined by the segmented linedefined by the L*a*b* values of 5 ppm and 10 ppm FD&C Blue No. 1 inaqueous solution.
 10. The method of claim 1, further comprising thesteps of: e) loading the selected one fraction or combination offractions containing separated anthocyanins on an ion exchange column;f) selectively separating the anthocyanins loaded on the ion exchangecolumn in step e) based on differences in charge and polarity of theanthocyanin molecules using a solvent of select pH; and g) selecting onefraction or a combination of fractions containing separated anthocyaninsseparated in step f) such that the separated anthocyanins selected instep g) when in an aqueous solution at a pH of 8.0 provides a maximumabsorbance of 620 nm to 635 nm.
 11. The method of claim 10, wherein theseparated anthocyanins selected in step f) in at least one concentrationin an aqueous solution at pH of 8.0 provides color characteristicshaving a ΔE value of 12 or less compared to the color characteristicsdefined by the segmented line defined by the L*a*b* values of 5 ppm and10 ppm FD&C Blue No. 1 in aqueous solution.
 12. The method of claim 1,wherein the step of selectively separating anthocyanins on the ionexchange column based on differences in charge and polarity of theanthocyanin molecules comprises the steps of (i) first using the solventof select pH which is a first eluting solvent to obtain a first fractionand (ii) using one or more subsequent eluting solvents of select pH toobtain the one fraction or combination of fractions, wherein eacheluting solvent is different, and the difference may be independentlyselected from the group of pH, solvent make-up and a combinationthereof.
 13. The method of claim 12, wherein the separated anthocyaninsin the one fraction or the combination of fractions, when in at leastone concentration in an aqueous solution at pH of 8.0 provides colorcharacteristics having a ΔE value of 12 or less compared to the colorcharacteristics defined by the segmented line defined by the L*a*b*values of 5 ppm and 10 ppm FD&C Blue No. 1 in aqueous solution.
 14. Themethod of claim 13, wherein the first eluting solvent is a mixture of anorganic solvent and water at a first concentration of organic solventand a subsequent eluting solvent is used that is a second mixture of anorganic solvent and water having a second concentration of organicsolvent, wherein the first concentration is different than the secondconcentration.
 15. The method of claim 12, wherein the first elutingsolvent has a select pH that is different than a select pH of thesubsequent eluting solvent.
 16. The method of claim 15, wherein theselect pH of the first eluting solvent is lower than the select pH ofthe subsequent eluting solvent.